Abstract
Background: Disseminated tumor cells (DTCs) in bone marrow (BM) occur in 30-40% of primary breast cancer patients. An impaired bone microenvironment may lead to reduced bone density and osteoporosis affecting the BM as a homing site for DTCs. The bone mineral density (BMD) and its correlation to DTC in BM was evaluated. Materials and Methods: One hundred and eighty-one women (70 premenopausal, 111 postmenopausal) underwent quantitative ultrasonometry before adjuvant chemotherapy. BM aspirates were analyzed by immunocytochemistry using the ACIS system (Chromavision) based on immunostaining. Results: DTCs were detected in 39% of the patients. Positive BM status correlated significantly with the nodal status. BMD was significantly reduced in the postmenopausal patients (p=0.003). Smaller tumors and higher BMD correlated significantly (p<0.014). Fifty percent of the patients with preclinical osteoporosis were BM positive, whereas 37% with normal or osteopenic BMD had DTCs. Conclusion: An impaired bone micro-environment as found in preclinical osteoporosis might be a homing site for DTCs.
Abbreviations: BM: Bone marrow; BMD: bone mineral density; BUA: broadband ultrasound attenuation; CK: cytokeratin; DEXA: dual-energy x-ray absorptiometry; DTCs: disseminated tumor cell(s); MRI: magnetic resonance imaging; QCT: quantitative computed tomography; QUS: quantitative ultrasonometry; RANKL: receptor activator of nuclear factor kappa B ligand; SI: stiffness index; SOS: speed of sound; n.s.: not significant.
The presence of disseminated tumor cells (DTCs) in bone marrow (BM) is a common phenomenon seen in primary breast cancer in 30-40% of patients. As demonstrated by a large pooled analysis of more than 4,700 patients, positive BM status at the time of diagnosis is an independent prognostic factor (1). Approximately 70-80% of the patients who will develop distant metastases present with DTCs at the time of surgery. The term DTC refers to any tumor cell that has left the primary tumor and reached an ectopic site. However, it is currently not predictable which of these cells will evolve into (micro-)metastases; the vast majority (99.9%) undergo apoptosis and very few are able to persist in secondary homing sites, such as BM (‘metastatic inefficiency’) (2). Why some patients are more likely to present with DTCs than others despite similar primary tumor characteristics remains to be explained. Recently, oncological research has focused increasingly not only on the cancer cell itself, but on complex interactions between malignant cells and their microenvironment. It is not yet clear which factors enable single tumor cells to reach the bone marrow and facilitate their persistence in this particular environment.
Bone tissue is a dynamic microenvironment where angiogenetic mechanisms, osteoclastic bone resorption and hormonal influences play an important role. An impaired bone microenvironment, in terms of high bone turnover, may lead to reduced bone density and osteoporosis. These changes may subsequently affect the function of BM as a homing site for DTCs. Micrometastatic breast cancer cells with the tendency to metastasize to bone may find a better microenvironment if osteoporosis already exists in the patient (3). Furthermore, the main agents used in bone-targeted therapy, bisphosphonates, suppress bone turnover and have thus a stabilizing effect on bone density. At the same time bisphosphonates show antitumorigenic activity in vivo as well as in vitro (4-7). Furthermore, these drugs may also act indirectly on cancer cells through microenvironmental changes using immunomodulatory and antiangiogenic mechanisms. Interestingly, while hormonal influences are a potential risk factor for developing breast cancer, they are closely related to bone metabolism by affecting osteoblasts and osteoclasts which leads to an altered microenvironment and subsequent osteoporosis. Whether (and how) microenvironmental changes in the bone affect the presence of DTCs remains under discussion.
Therefore, the aim of the present study was to evaluate bone density in primary breast cancer patients and its correlation to the presence of DTCs in BM.
Materials and Methods
Patient collective. A total of 181 primary breast cancer patients were included in the study. All the patients underwent primary breast surgery between 2000 and 2003 at the certified Breast Cancer Centre, University of Tuebingen, Germany. Exclusion criteria were neoadjuvant chemotherapy, other malignancies and age <18 years.
Quantitative ultrasonometry (QUS). For the sonographic measurements an Achilles device (Achilles Plus; GE-Lunar Corporation/Lunar GmbH, Cologne, Germany) was used, which consists of a transducer positioned each side of the heel. One transducer acts as transmitter and the other as receiver. Before measurement, the heel skin is cleaned with 70% isopropyl-alcohol, and it is then placed and fixed in the device. Acoustic coupling is accomplished by submerging the transducers and the patient's heel being measured in water at 33°C for 5 minutes. A surfactant solution improves acoustic coupling. Quality control with a standard phantom was performed weekly according to the manufacturer's instructions. Broadband ultrasound attenuation (BUA) was measured in decibels per megahertz and speed of sound (SOS) was measured in meters per second. BUA is caused by reflection and absorption of the scanned material when impulses of different frequencies are sent through the bone. SOS measures the time for an ultrasonographic signal to pass through the material. The passage time is shorter through cortical bone compared to trabecular bone. A more compact bone leads to a shorter penetration distance and consequently to a higher speed of transmission. The constant water temperature avoids effects on the calculation of BUA and SOS and minimizes confounding results. The BUA/SOS Index (stiffness index, SI) can be calculated from the SOS and BUA. The stiffness is the sum of standardized and scaled BUA and SOS values; a healthy 20-year-old female scores a value of 100. The SI compares the results with the SI values of young adults (T-Score) and adults in the same age group (Z-Score), and is used to reduce measurement errors due to variable water temperatures and heel widths. The lower the SI, the higher the fracture risk. Patients were stratified according to WHO classification into three groups: normal bone density (T-score >−1.0), osteopenia (T-score between −1.0 and −2.5) and preclinical osteoporosis (T-score <−2.5, no fractures). None of the included patients had manifest osteoporosis with fractures.
Detection of DTC in BM by immunocytochemistry. Using the technique of Jamshidi et al. technique (8), 10-20 ml of BM from each patient were aspirated intraoperatively from the anterior left or right iliac crest and processed within 24 hours for subsequent immunocytochemical detection of DTCs. Density centrifugation through Ficoll (density 1.077 g/ml; Biochrom, Berlin, Germany) was used to separate the BM samples. Mononuclear cells (MNC) were collected from the interphase layer, then washed with PBS and resuspended. Lysis of red blood cells was performed with lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH=7.2) when necessary. The cells (106 MNC/spot) were spun down onto a glass slide (Hettich cytocentrifuge, Tuttlingen, Germany) and air-dried overnight at room temperature. The slides were fixed in 4% neutral buffered formalin for 10 minutes and rinsed in PBS for the detection of cytokeratin (CK)-positive cells. Automatic immunostaining was performed on an Autostainer (DAKO Cytomation, Hamburg, Germany) using monoclonal mouse A45-B/B3 antibody for pancytokeratin (Mikromet, Munich, Germany) at a dilution of 1:400 and a DAKO-APAAP detection kit (DAKO Cytomation). The A45-B/B3 antibody is directed against cytokeratin epitopes including the CK heterodimers 8/18 and 8/19. The positive control was MCF-7 malignant breast cell line. The negative control was leucocytes of a healthy volunteer. A total of 2×106 cells were analyzed for each patient (two slides).The slides were automatically scanned using the ACIS™ imaging system (ChromaVision, Medical Systems Inc., San Juan, Capistrano, CA, USA) which is a computerized microscope with an image processing system optimized for the detection of rare cells in specimens. The characteristics of this system have been described in detail elsewhere (9). The criteria for the detection of DTCs were based on the recommendations of the European ISHAGE (International Society of Hematotherapy and Graft Engineering) Working group for standardization of tumor cell detection and the consensus statements (10, 11).
Ethical approval. Approval for the study was obtained from the local Ethics Committee of the University of Tuebingen, Germany (146/2004V) and was carried out according to the requirements of the declaration of Helsinki. Each patient signed an informed consent form beforehand.
Statistical analysis. The Chi-squared test was used to examine the relationship between the bone mass parameters obtained by QUS and the clinicopathological factors. Statistical analysis was performed by the SPSS computer program package (Version 11.5; IBM, Ehningen, Germany). P-values <0.05 were considered statistically significant.
Results
The basic patient characteristics of the study population are outlined in Table I. The age of the patients ranged from 31 to 84 years (median: 56 years). In 70 out of the 181 cases (39%), DTCs were detected in the BM at the time of surgery (Table I). The BM status correlated significantly with the presence of lymph node metastasis (p=0.026). No correlations were found between DTC detection and the other clinicopathological parameters. Figure 1 shows typical disseminated tumor cells from a breast cancer patient.
Bone mineral density (BMD) in breast cancer patients. The bone density parameters obtained by QUS are depicted in Tables II and III. Twenty-four patients had preclinical osteoporosis, with T-scores ranging from −2.50 to −5.19, whereas 60 patients were diagnosed with osteopenia (T-scores between −2.50 and −1.0), while 97 patients had normal bone density (T-score>−1.0). The bone density was significantly reduced in the postmenopausal patients (p=0.003). The median T-score in the premenopausal group was −0.26 (min. −3.33, max. 2.70) and in the postmenopausal group −1.12 (min. −5.19, max. 1.87). A statistically significant correlation between smaller tumor size and higher BMD was found (p<0.014). The median T-score for all the patients with T1 tumors was −0.69, for T2 tumors −1.56 and for T3/4 tumors −1.76. No correlation was observed between the bone density parameters and the other evaluated parameters. With regard to DTC presence, 12 out of the 24 (50%) of patients with preclinical osteoporosis were BM DTC-positive, whereas only 58 out of the 157 (37%) patients with normal or osteopenic bone density had DTCs in their BM (Table IV).
Presence of disseminated tumor cells (DTCs) in bone marrow and correlation with clinicopathological characteristics.
Discussion
The BM is a protective area for tumor cells where growth factors and cell adhesion molecules in the microenvironment can interact with tumor cells to enhance resistance to chemotherapy (12).
Immunocytochemistry. A cluster of disseminated tumor cells with typical cytomorphology and immunophenotype (positive cytokeratin staining, large nucleus, high nuclear to cytoplasmic ratio, nucleus partially covered by cytokeratin staining, granular nucleus).
Interestingly, survival benefit from the use of bisphosphonates depends on menopausal status, according to the recently reported results of the large AZURE (Adjuvant Zoledronic Acid to Reduce Recurrence) trial (13) in which women with established menopause had significantly longer disease-free survival (p=0.001). An estrogen-poor micro-environment seems to enhance the antitumor effects of bisphosphonates. Estrogens can have a mitogenic effect on breast tissue but estrogen deprivation is also likely to lead to significant bone loss (14) due to the effect on osteoblasts and osteoclasts and the formation of cytokines and mediators (15). A striking discrepancy in ER status between primary tumor and DTCs in BM was reported (16), which may lead to failure of conventional endocrine therapy. Isolated tumor cells might find optimal conditions in an estrogen-deprived microenvironment. Since bisphosphonates are able to target and eliminate DTCs from BM (17), it may be assumed that patients with low estrogen levels are more likely to benefit from bone-targeted therapy. Indeed, in the present study, 50% of the patients with preclinical osteoporosis presented with DTCs in their BM.
Therefore, oncology professionals need to intensify the routine assessment of osteoporosis risk in breast cancer patients. At present, it is an issue to find the optimal set up to monitor BMD and bone loss and select early-stage breast cancer patients who would benefit from bisphosphonates or receptor activator of nuclear factor kappa B ligand (RANKL) inhibitors to preserve BMD (18). Higher bone mass can be a marker for breast cancer risk (19), but a higher bone resorption is seen in patients with primary breast carcinoma that leads to a 5-fold increased incidence of vertebral fractures caused by osteoporosis (20). The optimal and cost-effective tool and interval for the measurement of a patient's bone density is not yet defined.
Bone mineral density and its correlation with clinicopathological characteristics. Bone density was classified according to the WHO guidelines into three groups: normal bone density (T-score >−1.0), osteopenia (T-score between −1.0 and −2.5) and preclinical osteoporosis (T-score <2.5, no fractures).
The projectional X-ray technique (dual-energy x-ray absorptiometry, DXA or DEXA), based on the absorption of x-rays in the vertebral column and in the femoral bone has been considered among the non-invasive methods of first choice but leads to a higher technical expenditure and radiation exposure. In recent years, ultrasound techniques have been established (21, 22) and are affordable, mobile and relatively simple to handle without radiation exposure for the patient. Transmission measurements at the heel provide the possibility to measure attenuation and SOS at the same time, which can be assessed separately or combined as SI (23, 24). The precision and accuracy of ultrasound techniques are comparable to the DEXA method.
Bone density parameters obtained by quantitative ultrasonometry.
Comparison of bone density and DTC detection in bone marrow (p=0.159).
Both reduced bone density and the presence of DTCs in BM are indicators of an impaired bone microenvironment. It remains unclear whether the higher bone mass in patients with smaller tumor size, as shown in the present population, was due to bone-protective estrogen levels as the patients' hormone levels were not investigated and compared. However, the findings were similar when tumor size and BMD were analyzed in the pre- and postmenopausal groups separately. The main objective of BMD monitoring and bone marrow assessment is to select the population that may benefit from bone-targeted therapies. Preventive bone targeted therapy could be started before adjuvant therapy to minimize the risk of osteoporosis-induced fractures (possibly as a consequence of adjuvant therapy). The treatment of therapy-induced osteoporosis is recommended based on the monitoring of BMD and osteoporosis risk factors (25-27). A risk collective of patients that is identified by the presence of DTCs or ultrasound of the heel might benefit from further BMD-preserving therapy options such as bisphosphonates.
Conclusion
Patients with preclinical osteoporosis are more likely to present with DTCs in their BM than patients with normal/osteopenic bone density. Bone density monitoring in combination with the detection of DTC might be a strategy to define a population that may benefit most from bone-targeted therapies.
Acknowledgements
The Authors thank Miss Carmen Mack for the measurement of the patients' BMD and for the collecting of data.
Footnotes
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Competing Interests
The Authors declare that they have no conflicts of interests.
- Received August 26, 2011.
- Revision received October 26, 2011.
- Accepted October 28, 2011.
- Copyright© 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
